The majority of chemotherapeutic agents used to treat cancer exhibit serious toxicity, resulting in undesired side effects for patients and reducing efficacy by limiting the doses that can be safely administered. Similarly, many of the therapeutics used to treat infectious diseases, including parasitic diseases, confer undesirable side effects. It would be preferable if such agents could be administered in a prodrug form that masked the inherent toxicity of the agent from irrelevant, non-diseased tissues, and yet released the fully active drug species at the desired site of action. Such a technology would have the potential to increase the therapeutic window of a variety of drugs, possibly allowing them to be used safely at a more efficacious dose, and with reduced incidence of undesired side-effects for the patient.
In normal cells and tissues, iron remains sequestered in forms that are non-toxic to the cell, bound to the iron carrying protein transferrin for example, or bound as heme within hemoglobin. Diseased tissues and cells, on the other hand, can contain higher than normal concentrations of iron. Many neoplastic cells for example over-express the transferrin receptor to increase their uptake of iron. Increased iron uptake has been proposed to explain the increased toxicity that iron-dependent endoperoxides like artemisinin exhibit towards cancer cell lines as compared to normal cells (Efferth, T. Drug Resistance Updates, 2005, 8:85-97). In one study, the expression level of the transferrin receptor was shown to correlate with the cytotoxicity of an artemisinin derivative towards HeLa cells (see for example Disbrow, G. L., et al Cancer Research, 2005, 65, 10854-10861). Artemisinin and its derivatives are believed to exert their cytotoxic effect via reaction with FeII and the resulting generation of reactive oxygen and carbon centered radical species. The cytotoxicity of artemisinin derivatives towards leukemia, astrocytoma, and breast cancer cell lines can be potentiated by the addition of exogenous FeII salts or transferrin (Efferth, T. et al Free Radical Biology & Medicine, 2004, 37, 998-1009; Singh, N. P. et al Life Sciences, 2001, 70, 49-56). U.S. Pat. No. 5,578,637 describes the use of an endoperoxide moiety (i.e., an artemisinin) to kill cancer cells under conditions that enhance intracellular iron concentrations. None of these prior works teach or suggest how higher than normal concentrations of iron in such cells could be exploited for selective delivery of a drug species via an iron-sensitive prodrug moiety.
The blood-scavenging parasites responsible for diseases such as malaria and schistosomiasis also possess biological compartments rich in ferrous iron. In malaria parasites, unbound heme is generated in the parasite digestive vacuole where hemoglobin is degraded by a number of proteases (See Rosenthal, P. J. in Protease and hemoglobin degradation. Molecular Approaches to Malaria, 2005: p. 311-326). Hence, while the concentration of unbound, ferrous iron is vanishingly small in human plasma (˜10−16M), significant quantities of ferrous iron are present within malaria parasites (see Robert, A. et al Coordination Chemistry Reviews, 2005, 249, p. 1927-1936). The antimalarial drug artemisinin and its related synthetic derivatives are thought to confer their antiparasitic effect via reaction with ferrous iron and the resulting generation of reactive oxygen and carbon centered radical species. An excess of iron, and ferrous iron in particular, is therefore a distinguishing characteristic of many neoplastic cells and pathogenic parasites.
Among synthetic endoperoxide ring systems, the iron reactivity of 1,2,4-trioxolanes has been extensively studied in vitro using model systems (see Creek, D. J. et al, J. Pharm. Sci. 2007, 96, 2945-2956). As shown in FIG. 1, exposure of trioxolane A to iron(II) acetate, leads primarily to the formation of cyclohexanone (E) and the adamantane-derived lactone D, presumably via intermediates B and C (Tang, Y. et al J. Org. Chem. 2005, 70, 5103-5110). O'Neill and co-workers have devised endoperoxide systems in which the carbonyl compound formed upon reaction with iron is itself a chalcone species with antimalarial activity (O'Neill et al, Angewandte Chemie, International Edition, 2004, 43, 4193-4197 and Org. Lett. 2004, 6, 3035-3038). Although these systems can be viewed as prodrugs of chalcones, this work in no way teaches or suggests how the approach might be applied to a wide variety of drug species (i.e., drugs other than chalcones). Further, attachment of a blocking moiety (“pro” moiety) at a carbonyl function as reported in this work is much less desirable and is of less utility than attachment at an amine or alcohol function.
The use of prodrugs to confer improved properties such as increased bioavailability or aqueous solubility is a well established concept in the art of pharmaceutical research. These standard approaches rely on the action of serum esterases or phosphatases to remove the blocking pro moiety and thereby liberate the drug species. The attachment of a cytotoxic agent to a targeting moiety such as a protein or antibody via an acid-labile linker moiety is another known prodrug approach, intended to deliver a drug moiety to a specific cell or tissue. See U.S. Pat. No. 5,306,809. Acid labile linker moieties have also been used to attach drug species to biopolymers or antibodies where the intention is that the lower pH of the diseased tissue serves to trigger release of the drug moiety. See U.S. Pat. Nos. 4,631,190; 4,997,913; 5,140,013. The use of a masked retro-Michael linker has been employed in a targeted prodrug system involving biochemical oxidation as the trigger for drug release (Huttunen, et al, 2007, Pharm. Res., 24:679-687). In this system, oxidation of a cyclic phosphate prodrug by CYP-450 enzymes exposes a retro-Michael substrate that undergoes spontaneous β-elimination to release phosphorylated drug species. None of these prior studies teach or suggest how one might mask the carbonyl function of a retro-Michael substrate as a trioxane or trioxolane ring, and attach such moiety to a drug species, thereby producing a prodrug that effectively releases active drug only in cells or tissues having ferrous iron concentrations above the physiological norm.